Human gastric inhibitory peptide (active) ELISA kit instruction manual

2021-08-04 23:43:09

Human gastric inhibitory peptide ( active ) ELISA test kit <br>Japan IBL imported, article number: 27201
Japan's IBL China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you

Introduction to Preface <br>Incretin is a class of gut hormones. When the human body eats, it can induce the release of insulin from islet β cells and inhibit the release of glucagon.
GIP is a typical GLP-1 incretin isolated from the intestinal mucosa in 1970, called gastric inhibitory peptide, and later renamed as glucose-dependent insulinotropic peptide. It is composed of 43 amino acids. The chain peptide, belonging to the family of secretin and glucagon, has a molecular weight of 5,100 and is produced by K cells of the small intestinal mucosa. It has been reported that GIP receptors are expressed in a wide variety of cells (such as pancreatic beta cells, adipocytes, human osteoblasts) and play a vital role in the storage of nutrients in the body's cells, while regulating GIP signals. Improve insulin resistance syndrome.
Active GIP (1-42) in the blood can be rapidly inactivated to GIP by the action of dipeptidyl peptidase IV (DPP-IV) (3-42)
This ELISA test kit is only used for the detection of active gastric inhibitory peptide (1-42).
Principle <br>This kit is based on the principle of sandwich method, using two specific antibodies, after washing the plate to add TMB substrate liquid color development, the intensity of color development is proportional to the amount of human active gastric inhibitory peptide in the sample.

examination range
1.6 - 100 pg/mL (0.3 - 20 pmol/L)

Intended use <br>For scientific research only This IBL kit can be used for quantitative detection of human active gastric inhibitory peptide (GIP) in EDTA-plasma. Samples need to be added with dipeptidyl peptidase IV (DPP-IV) inhibitor Or use a specific method to ensure the integrity of the inhibitory peptide.
Kit component
1 pre-coated plate: anti-GIP (C) rabbit IgG, affinity purification 96T
2 enzyme labeled antibody:
(30 times concentrated) HRP-labeled anti-GIP (N) (6A1A) mouse IgG, affinity purification 0.4mL x 1
3 Standard: Human GIP (1-42) 0.5mL x 2
4 EIA buffer: 1% BSA, 0.05% Tween 20 BPS 30mL x 1
5 labeled antibody dilution: 1% BSA, 0.05% Tween 20 BPS 12mL x 1
6 Reagent: TMB substrate liquid 15mL x 1
7 Stop solution: 1N sulfuric acid 12mL x 1
8 Concentrated washing solution:
(40 times concentrated) with 1% BSA, 0.05% Tween 20 BPS 50mL x 1

Instructions
1 equipment required for the experiment ( but not provided in the kit )
Microplate reader Micropipette and its nozzles Measuring cylinder and beaker Deionized water Refrigerator (4°C) Coordinate paper (log/log)
Absorbent paper test tube (for standard dilution)
Incubation box (37 ° C ± 1 ° C)
Wash bottle (for washing)
Disposable reagent tubes (for concentrated enzyme-labeled antibodies and developers)
2 preparation
  1. Preparation of washing liquid
The washing solution of the kit is a 40-fold concentrated solution. Dilute 50 mL of the concentrated washing solution with 1950 mL to prepare a 2000 mL ready-to-use diluted washing solution. It can be stored in the refrigerator for 2 weeks.
  1. Preparation of enzyme-labeled antibodies
The labeled antibody in the kit is a 30-fold concentrate diluted with the labeled antibody dilution according to the required amount.
Example:
If you use one plate (8 wells), you need 800 μL of labeled antibody. Dilute 30 μL of the concentrate with 870 μL of labeled antibody dilution and mix well. Add 100 μL to each well.
Concentrated enzyme-labeled antibodies need to be diluted before they can be used in experiments. <br>The remaining diluted labeled antibody solution needs to be sealed and stored at 4 °C.
  1. Standard preparation
Add 0.5 mL of deionized water to the bottled standard to make a 200 μg/mL human GIP standard solution.
  1. Dilution of standards
Prepare 8 tubes, and the respective Each added 230 μ L EIA buffer was diluted to the following concentrations, as shown below steps:
Test tube 1 100 pg/mL
Tube 2 50 pg/mL
Tube 3 25 pg/mL
Tube 4 12.5 pg/mL
Tube 5 6.3 pg/mL
Tube 6 3.1 pg/mL
Tube 7 1.6 pg/mL
Tube 8 0 pg/mL (sample blank)
Pipette 230 ul of standard solution into the No. 1 tube and mix, then pipette 230 ul from the No. 1 tube and add the No. 2 tube to mix. As shown in the figure below, dilute according to this rule. The No. 8 tube is 0 pg/mL as the sample blank.

  1. Sample dilution
If the sample needs to be diluted, it must be diluted with EIA buffer. If the concentration range of human GIP in the sample is unknown, prepare several different dilutions of sample solution to determine the best detection concentration.
3 Experimental Procedures <br> Before use, all reagents should be equilibrated to room temperature for about 30 minutes, thoroughly mixed to ensure that the reagent quality is unchanged, and the standard curve and sample detection must be performed simultaneously.

Reagent Sample to be tested Standard Sample blank Reagent blank
Test sample 100ul Dilution standard (1-7 tubes) 100ul EIA buffer (8th tube) 100ul EIA buffer 100ul
Cover plate, incubated at 37 ° C for 60 min
Wash the plate 4 times
Enzyme-labeled antibody 100ul 100ul 100ul -
Cover plate, incubated at 4 ° C for 60 min
Wash the plate 5 times
Reagent 100ul 100ul 100ul 100ul
Incubate at room temperature for 30 min in the dark
Stop solution 100ul 100ul 100ul 100ul
After adding the stop solution, read the OD value at 450 nm within 30 min.
1) Set a reagent blank and add 100 ul EIA buffer to the corresponding microwell.
2) Determine the sample blank control well, test the sample well and standard well, and add 100ul sample control (8th tube), test sample and diluted standard (1-7 tube) to the corresponding microwell. in.
3) Cover plate, incubated at 37 ° C for 60 min
4) Wash the plate 4 times with a washing bottle or automatic washing machine as follows: Pour the reaction solution in the well, fill each well with the diluted washing solution, and then discard the waste liquid. Repeat the plate washing 4 times . On the absorbent paper Pat dry to remove residual droplets.
5) In addition to the reagent blank control well, add 100 ul of enzyme-labeled antibody to each well.
6) Cover plate, incubate for 60 min at 4 °C
7) Wash the plate 5 times according to the above step 4)
8) Use a disposable tube to take the required amount of developer, add 100ul of each developer to the microwell. To avoid contamination, do not refill the reagent in the reagent bottle.
9) Incubate at room temperature for 30 minutes in the dark, and the color of the solution will turn blue after adding the color developer.
10) Add 100 ul of stop solution to each well and the color of the solution will turn yellow.
11) Wipe off the stains or water droplets at the bottom of the microplate to ensure that no bubbles are generated in the solution in the micropores. Read the results at 450nm after 30 minutes of adding the stop solution.
pay attention
  1. As soon as the sample is collected, it will be tested as soon as possible. If necessary, store the sample frozen, but avoid repeated freezing and thawing. Thaw the sample to room temperature before testing.
  2. If the sample needs to be diluted, dilute with EIA buffer
  3. Recommended for double detection
  4. Keep the sample in a neutral pH range. Contamination of organic solvents can affect the results of the experiment.
  5. Wash the plate only with the wash solution provided with this kit. Insufficient wash plate will result in erroneous results
  6. When pat dry on absorbent paper to remove residual droplets, do not scratch the inner wall of the hole with absorbent paper.
  7. The display agent should be stored away from light and avoid contact with metallic substances
  8. After adding the stop solution, the test results should be read within 30 min.

Results Calculations <br>All experimental data (including standards, OD values ​​of test samples) should be subtracted from the OD value of the sample blanks.
In the log-log coordinate paper, the corrected OD value is the ordinate, and the corresponding concentration is the abscissa, and the standard curve is established. The sample directly obtains the concentration value on the standard curve.
Standard curve demonstration


The typical standard curve shown above is for demonstration purposes only and cannot be used for the acquisition of test data. The standard curve should be established for each experiment.

General matters
1 All reagents were stored at 2 - 8 ° C. The reagents were equilibrated to room temperature (about 30 minutes) before the experiment.
The 2 bottle standard is lyophilized, so carefully open the cap
3 Stop liquid is a strong acid substance. Avoid contact with skin and skin when using.
4 Rinse with water before discarding used materials
5 Concentrated enzyme-labeled antibody, EIA buffer and concentrated washing solution may precipitate, but this will not affect the experiment.
6 Wash hands after using reagents
7 Do not mix reagents of different batches
8 Do not use expired reagents
9 kits are for research use only and cannot be used for clinical diagnosis.


Storage and expiration date <br>Storage conditions: 2 - 8 °C
See the kit packaging for the expiration date.
This translation is for reference only, please refer to the original for details.

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